Variant of LAV viruses

ABSTRACT

A variant of a LAV virus, designated LAV ELI  and capable of causing AIDS. The cDNA and antigens of the LAV ELI  virus can be used for the diagnosis of AIDS and pre-AIDS.

This is a continuation of application Ser. No. 08/423,477, filed Apr.19, 1995, which is a division of application Ser. No. 07/656,796, filedFeb. 19, 1991 now U.S. Pat. No. 5,869,631, which is a division ofapplication Ser. No. 07/038,332, filed Apr. 13, 1987, now U.S. Pat. No.5,034,511, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a virus capable of inducinglymphadenopathies (hereinafter “LAS”) and acquired immuno-depressivesyndromes (hereinafter “AIDS”), to antigens of this virus, particularlyin a purified form, and to a process for producing these antigens,particularly antigens of the envelope of this virus. The invention alsorelates to polypeptides, whether glycosylated or not, produced by thevirus and to DNA sequences which code for such polypeptides. Theinvention further relates to cloned DNA sequences hybridizable togenomic RNA and DNA of the lymphadenopathy associated virus (hereinafter“LAV”) of this invention and to processes for their preparation andtheir use. The invention still further relates to a stable probeincluding a DNA sequence which can be used for the detection of the LAVvirus of this invention or related viruses or DNA proviruses in anymedium, particularly biological, and in samples containing any of them.

An important genetic polymorphism has been recognized for the humanretrovirus which is the cause of AIDS and other diseases like LAS,AIDS-related complex (hereinafter “ARC”) and probably someencephalopathies (for review, see Weiss, 1984). Indeed all of theisolates, analyzed until now, have had distinct restriction maps, eventhose recovered at the same place and time [Benn et al., 1985].Identical restriction maps have only been observed for the first twoisolates which were designated LAV [Alizon et al., 1984] and humanT-cell lymphotropic virus type 3 (hereinafter “HTLV-3”) [Hahn et al.,1984] and which appear to be exceptions. The genetic polymorphism of theAIDS virus was better assessed after the determination of the completenucleotide sequence of LAV [Wain-Hobson et al., 1985], HTLV-3 [Ratner etal., 1985; Muesing et al., 1985] and a third isolate designatedAIDS-associated retrovirus (hereinafter “ARV 2”) [Sanchez-Pescador etal., 1985]. In particular, it appeared that, besides the nucleic acidvariations responsible for the restriction map polymorphism, isolatescould differ significantly at the protein level, especially in theenvelope (up to 13% of difference between ARV and LAV), by both aminoacids substitutions and reciprocal insertions-deletions [Rabson andMartin, 1985].

Nevertheless, such differences did not go so far as to destroy theimmunological similarity of such isolates as evidenced by thecapabilities of their similar proteins, (e.g., core proteins of similarnature, such as the p25 proteins, or similar envelope glycoproteins,such as the 110–120 kD glycoproteins) to immunologically cross-react.Accordingly, the proteins of any of said LAV viruses can be used for thein vitro detection of antibodies induced in vivo and present inbiological fluids obtained from individuals infected with the other LAVvariants. Therefore, these viruses are grouped together as a class ofLAV viruses (hereinafter “LAV-1 viruses”).

SUMMARY OF THE INVENTION

In accordance with this invention, a new virus has been discovered thatis responsible for diseases clinically related to AIDS and that can beclassified as a LAV-1 virus but that differs genetically from knownLAV-1 viruses to a much larger extent than the known LAV-1 virusesdiffer from each other. The new virus is basically characterized by thecDNA sequence which is shown in FIGS. 7A to 7I, and this new virus ishereinafter generally referred to as “LAV_(ELI)”.

Also in accordance with this invention, variants of the new virus areprovided. The RNAs of these variants and the related cDNAs derived fromsaid RNAs are hybridizable to corresponding parts of the cDNA ofLAV_(ELI). The DNA of the new virus also is provided, as well as DNAfragments derived therefrom hybridizable with the genomic RNA ofLAV_(ELI) such DNA and DNA fragments particularly consisting of the cDNAor cDNA fragments of LAV_(ELI) or of recombinant DNAs containing suchcDNA or cDNA fragments.

DNA recombinants containing the DNA or DNA fragments of LAV_(ELI) or itsvariants are also provided. It is of course understood that fragmentswhich would include some deletions or mutations which would notsubstantially alter their capability of also hybridizing with theretroviral genome of LAV_(ELI) are to be considered as forming obviousequivalents of the DNA or DNA fragments referred to hereinabove.

Cloned probes are further provided which can be made starting from anyDNA fragment according to the invention, as are recombinant DNAscontaining such fragments, particularly any plasmids amplifiable inprocaryotic or eucaryotic cells and carrying said fragments. Usingcloned DNA containing a DNA fragment of LAV_(ELI) as a molecularhybridization probe—either by marking with radionucleotides or withfluorescent reagents—LAV virion RNA may be detected directly, forexample, in blood, body fluids and blood products (e.g., inantihemophylic factors such as Factor VIII concentrates). A suitablemethod for achieving such detection comprises immobilizing LAV_(ELI) ona support (e.g., a nitrocellulose filter), disrupting the virion andhybridizing with a labelled (radiolabelled or “cold” fluorescent- orenzyme-labelled probe. Such an approach has already been developed forHepatitis B virus in peripheral blood (according to Scotto J. et al.Hepatology (1983), 3, 379–384).

Probes according to the invention can also be used for rapid screeningof genomic DNA derived from the tissue of patients with LAV relatedsymptoms to see if the proviral DNA or RNA present in their tissues isrelated to LAV_(ELI). A method which can be used for such screeningcomprises the following steps: extraction of DNA from tissue,restriction enzyme cleavage of said DNA, electrophoresis of thefragments and Southern blotting of genomic DNA from tissues andsubsequent hybridization with labelled cloned LAV proviral DNA.Hybridization in situ can also be used. Lymphatic fluids and tissues andother non-lymphatic tissues of humans, primates and other mammalianspecies can also be screened to see if other evolutionary relatedretroviruses exist. The methods referred to hereinabove can be used,although hybridization and washings would be done under non-stringentconditions.

The DNA according to the invention can be used also for achieving theexpression of LAV viral antigens for diagnostic purposes, as well as forthe production of a vaccine against LAV. Fragments of particularadvantage in that respect will be discussed later. The methods which canbe used are multifold:

a) DNA can be transfected into mammalian cells with appropriateselection markers by a variety of techniques, such as calcium phosphateprecipitation, polyethylene glycol, protoplast-fusion, etc.

b) DNA fragments corresponding to genes can be cloned into expressionvectors for E. coli, yeast or mammalian cells and the resultant proteinspurified.

c) The provival DNA can be “shot-gunned” (fragmented) into procaryoticexpression vectors to generate fusion polypeptides.

Recombinants, producing antigenically competent fusion proteins, can beidentified by simply screening the recombinants with antibodies againstLAV_(ELI) antigens. Particular reference in this respect is made tothose portions of the genome of LAV_(ELI) which, in the figures, areshown to belong to open reading frames and which encode the productshaving the polypeptidic backbones shown.

Different polypeptides which appear in FIGS. 7A to 7I are still furtherprovided. Methods disclosed in European application O 178 978 and in PCTapplication PCT/EP 85/00548, filed Oct. 18, 1985, are applicable for theproduction of such peptides from LAV_(ELI). In this regard, polypeptidesare provided containing sequences in common with polypeptides comprisingantigenic determinants included in the proteins encoded and expressed bythe LAV_(ELI) genome. Means are also provided for the detection ofproteins of LAV_(ELI) particularly for the diagnosis of AIDS or pre-AIDSor, to the contrary, for the detection of antibodies against LAV_(ELI)or its proteins, particularly in patients afflicted with AIDS orpre-AIDS or more generally in asymtomatic carriers and in blood-relatedproducts. Further provided are immunogenic polypeptides and moreparticularly protective polypeptides for use in the preparation ofvaccine compositions against AIDS or related syndroms.

Yet further provided are polypeptide fragments having lower molecularweights and having peptide sequences or fragments in common with thoseshown in FIGS. 7A to 7I. Fragments of smaller sizes can be obtained byresorting to known techniques, for instance, by cleaving the originallarger polypeptide by enzymes capable of cleaving it at specific sites.By way of examples may be mentioned the enzyme of Staphylococcyus aureusV8, α-chymotrypsine, “mouse sub-maxillary gland protease” marketed bythe Boehringer company, Vibrio alginolyticus chemovar iophaguscollagenase, which specifically recognizes the peptides Gly-Pro,Gly-Ala, etc.

Other features of this invention will appear in the following disclosureof data obtained starting from LAV_(ELI), in relation to the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide comparative restriction maps of the genomas ofLAV_(ELI) as compared to LAV_(MAL) (Applicants' related new LAV viruswhich is the subject of their copending application, filed herewith) andLAV_(BRU) (a known LAV isolate deposited at the Collection Nationale desCultures de Micro-organismes (hereinafter “CNCM”) of the PasteurInstitute, Paris, France under No. I-232 on Jul. 15, 1983);

FIG. 2 shows comparative maps setting forth the relative positions ofthe open reading frames of the above genomas;

FIGS. 3A–3F (also designated generally hereinafter “FIG. 3”) indicatethe relative correspondence between the proteins (or glycoproteins)encoded by the open reading frames, whereby amino acid residues ofprotein sequences of LAV_(ELI) are in vertical alignment withcorresponding amino acid residues (numbered) of corresponding orhomologous proteins or glycoproteins of LAV_(BRU), as well as LAV_(MAL)and ARV 2;

FIGS. 4A–4B (also designated generally hereinafter “FIG. 4”) providetables quantitating the sequence divergence between homologous proteinsof LAV_(BRU), LAV_(ELI) and LAV_(MAL);

FIG. 5 shows diagrammatically the degree of divergence of the differentvirus envelope proteins;

FIGS. 6A and 6B (“FIG. 6” when consulted together) render apparent thedirect repeats which appear in the proteins of the different AIDS virusisolates.

FIGS. 7A–7I show the full nucleotidic sequences of LAV_(ELI).

DETAILED DESCRIPTION OF THE INVENTION

Characterization and Molecular Cloning of an African Isolate.

The different AIDS virus isolates concerned are designated by threeletters of the patients name, LAV_(BRU) referring to the prototype AIDSvirus isolated in 1983 from a French homosexual patient with LAS andthought to have been infected in the USA in the preceding years[Barré-Sinoussi et al., 1983]. LAV_(ELI) was recovered in 1983 from a24-year old woman with AIDS from Zaire. Related LAV_(MAL) was recoveredin 1985 from a 7-year old boy from Zaire. Recovery and purification ofthe LAV_(ELI) virus were performed according to the method disclosed inEuropean Patent Application 84 401834/138667 filed on Sep. 9, 1984.

LAV_(ELI) is indistinguishable from the previously characterizedisolates by its structural and biological properties in vitro. Virusmetabolic labelling and immune precipitation by patient ELI sera, aswell as reference sera, showed that the proteins of LAV_(ELI) had thesame molecular weight (hereinafter “MW”) as, and cross-reactedimmunologically with those of, prototype AIDS virus (data not shown) ofthe LAV-1 class.

Reference is again made to European Application 178 978 andInternational Application PCT/EP 85/00548 as concerns the purification,mapping and sequencing procedures used herein. See also the discussionunder the headings “Experimental Procedures” and “Significance of theFigures” hereinafter.

Primary restriction enzyme analysis of LAV_(ELI) genome was done bysouthern blot with total DNA derived from acutely infected lymphocytes,using cloned LAV_(BRU) complete genome as probe. Overallcross-hybridization was observed under stringent conditions, but therestriction profile of the Zairian isolate was clearly different. Phagelambda clones carrying the complete viral genetic information wereobtained and further characterized by restriction mapping and nucleotidesequence analysis. A Clone (hereinafter “E-H12”) was derived fromLAV_(ELI) infected cells and contained an integrated provirus with 5′flanking cellular sequences but a truncated 3′ long terminal repeat(hereinafter “LTR”).

FIG. 1B gives a comparaison of the restriction maps derived from thenucleotide sequences of LAV_(ELI), LAV_(MAL) and prototype LAV_(BRU), aswell as from three other Zairian isolates (hereinafter “Z1”, “Z2”, and“Z3” respectively) previously mapped for seven restriction enzymes [Bennet al., 1985]. Despite this limited number, all of the profiles areclearly different (out of the 23 sites making up the map of LAV_(BRU),only seven are present in all six maps presented), confirming thegenetic polymorphism of the AIDS virus. No obvious relationship isapparent between the five Zairian maps, and all of their common sitesare also found in LAV_(BRU).

Conservation of the Genetic Organization.

The genetic organization of LAV_(ELI) as deduced from the completenucleotide sequences of its cloned genome is identical to that found inother isolates, i.e., 5′gag-pol-central region-env-F3′. Most noticeableis the conservation of the “central region” (FIG. 2), located betweenthe pol and env genes, which is composed of a series of overlapping openreading frames (hereinafter “orf”) previously designated Q, R, S, T, andU in the ovine lentivirus visna [Sonigo et al., 1985]. The product oforf S (also designated “tat”) is implicated in the transactivation ofvirus expression [Sodroski et al., 1985; Arya et al., 1985]; thebiological role of the product of orf Q (also designated “sor” or “orfA”) is still unknown [Lee et al., 1986; Kang et al., 1986]. Of the threeother orfs, R, T, and U, only orf R is likely to be a seventh viralgene, for the following reasons: the exact conservation of its relativeposition with respect to Q and S (FIG. 2), the ponstant presence of apossible splice acceptor and of a consensus AUG initiator codon, itssimilar codon usage with respect to viral genes, and finally the factthat the variation of its protein sequence within the different isolatesis comparable to that of gag, pol and Q (see FIG. 4).

Also conserved are the sizes of the U3, R and U5 elements of the LTR(data not shown), the location and sequence of their regulatory elementssuch as TATA box and AATAAA polyadenylation signal, and their flankingsequences, i.e., primer binding site (hereinafter “PBS”) complementaryto 3′ end of tRNA^(LYS) and polypurine tract (hereinafter “PPT”). Mostof the genetic variability within the LTR is located in the 5′ half ofU3 (which encodes a part of orf F) while the 3′ end of U3 and R, whichcarry most of the cis-acting regulatory elements, promoter, enhancer andtrans-activating factor receptor [Rosen et al., 1985], as well as the U5element, are well-conserved.

Overall, it clearly appears that this Zairian isolate, LAV_(ELI), is thesame type of retrovirus as the previously sequenced isolates of Americanor European origin.

Variability of the Viral Proteins.

Despite their identical genetic organization, the LAV_(ELI) andLAV_(MAL) shows substantial differences in the primary structure oftheir proteins. The amino acid sequences of LAV_(ELI) and LAV_(MAL)proteins are presented in FIGS. 3A–3F, aligned with those of LAV_(BRU)and ARV 2. Their divergence was quantified as the percentage of aminoacids substitutions in two-by-two alignments (FIG. 4). The number ofinsertions and deletions that had to be introduced in each of thesealignments has also been scored.

Three general observations can be made. First, the protein sequences ofthe LAV_(ELI) and LAV_(MAL) are more divergent from LAV_(BRU) than arethose of HTLV-3 and ARV 2 (FIG. 4A); similar results are obtained if ARV2 is taken as reference (not shown). The range of genetic polymorphismbetween isolates of the AIDS virus is considerably greater thanpreviously observed. Second, our two sequences confirm that the envelopeis more variable than the gag and pol genes. Here again, the relativelysmall difference observed between the env of LAV_(BRU) and HTLV-3appears as an exception. Third, the mutual divergence of the LAV_(ELI)and LAV_(MAL) (FIG. 4B) is comparable to that between LAV_(BRU) andeither of them; as far as we can extrapolate from only three sequencedisolates from the USA and Europe and two (LAV_(ELI) and LAV_(MAL)) fromAfrica, this is indicative of a wider evolution of the AIDS virus inAfrica.

-   gag and pol: Their greater degree of conservation compared to the    envelope is consistent with their encoding important structural or    enzymatic activities. Of the three mature gag proteins, the p25    which was the first recognized immunogenic protein of LAV    [Barré-Sinoussi et al., 1983] is also the better conserved (FIG. 3).    In gag and pol, differences between isolates are principally due to    point mutations, and only a small number of insertional or    deletional events is observed. Among these, we must note the    presence in the overlapping part of gag and pol of LAV_(BRU) of an    insertion of 12 amino acids (AA) which is encoded by the second copy    of a 36 bp direct repeat present only in this isolate and in HTLV-3.    This duplication was omitted because of a computing error in the    published sequence of LAV_(BRU) (position 1712, Wain-Hobson et    al., 1985) but was indeed present in the HTLV-3 sequences [Ratner et    al., 1985; Muesing et al., 1985].-   env: Three segments can be distinguished in the envelope    glycoprotein precursor [Allan et al., 1985; Montagnier et al., 1985;    DiMarzoVeronese et al., 1985]. The first is the signal peptide    (positions 1–33 in FIG. 3), and its sequence appears as variable;    the second segment (pos. 34–530) forms the outer membrane protein    (hereinafter “OMP” or “gp110”) and carries most of the genetic    variations, and in particular almost all of the numerous reciprocal    insertions and deletions; the third segment (531–877) is separated    from the OMP by a potential cleavage site following a constant basic    stretch (Arg-Glu-Lys-Arg) and forms the transmembrane protein    (hereinafter “TMP” or “gp 41”) responsible for the anchorage of the    envelope glycoprotein in the cellular membrane. A better    conservation of the TMP than the OMP has also been observed between    the different murine leukemia viruses (hereinafter “MLV”) [Koch et    al., 1983] and could be due to structural constraints.

From the alignment of FIG. 3 and the graphical representation of theenvelope variability shown in FIG. 5, we clearly see the existence ofconserved domains, with little or no genetic variation, andhypervariable domains, in which even the alignment of the differentsequences is very difficult, because of the existence of a large numberof mutations and of reciprocal insertions and deletions. We have notincluded the sequence of the envelope of the HTLV-3 isolate since it soclose to that of LAV_(BRU) (cf. FIG. 4), even in the hypervariabledomains, that it did not add anything to the analysis. While thisgraphical representation will be refined by more sequence data, thegeneral profile is already apparent, with three hypervariable domains(Hyl, 2 and 3) all being located in the OMP and separated by threewell-conserved stretches (residues 37–130, 211–289, and 488–530 of FIG.3 alignment) probably associated with important biological functions.

In spite of the extreme genetic variability, the folding pattern of theenvelope glycoprotein is probably constant. Indeed the position ofvirtually all of the cysteine residues is conserved within the differentisolates (FIGS. 3 and 5), and the only three variable cysteines falleither in the signal peptide or in the very C-terminal part of the TMP.The hypervariable domains of the OMP are bounded by conserved cysteines,suggesting that they may represent loops attached to the common foldingpattern. Also the calculated hydropathic profiles [Kyte and Doolittle,1982] of the different envelope proteins are remarkably conserved (notshown).

About half of the potential N-glycosylation sites, Asn-X-Ser/Thr, foundin the envelopes of the Zairian isolates map to the same positions inLAV_(BRU) (17/26 for LAV_(ELI) and 17/28 for LAV_(MAL)). The other sitesappear to fall within variable domains of env, suggesting the existenceof differences in the extent of envelope glycosylation between differentisolates.

Other viral proteins: Of the three other identified viral proteins, thep27 encoded by orf F, 3′ of env [Allan et al., 1985b] is the mostvariable (FIG. 4). The proteins encoded by orfs Q and S of the centralregion are remarkable by their absence of insertions/deletions.Surprisingly, a high frequency of amino acids substitutions, comparableto that observed in env, is found for the product of orf S(trans-activating factor). On the other hand, the protein encoded by orfQ is no more variable than gag. Also noticeable is the lower variationof the proteins encoded by the central regions of LAV_(ELI) andLAV_(MAL).

With the availability of the complete nucleotide sequence from fiveindependent isolates, some general features of the AIDS virus' geneticvariability are now emerging. Firstly, its principal cause is pointmutations which very often result in amino acid substitutions and whichare more frequent in the 3′ part of the genome (orf S, env and orf F).Like all RNA viruses, the retroviruses are thought to be highly subjectto mutations caused by errors of the RNA polymerases during theirreplication, since there is no proofreading, of this step [Holland etal., 1982; Steinhauer and Holland, 1986].

Another source of genetic diversity is insertions/deletions. From theFIG. 3 alignments, insertional events seem to be implicated in most ofthe cases, since otherwise deletions should have occurred in independentisolates at precisely the same locations. Furthermore, upon analyzingthese insertions, we have observed that they most often represent one ofthe two copies of a direct repeat (FIG. 6). Some are perfectly conservedlike the 36 bp repeat in the gag-pol overlap of LAV_(BRU) (FIG. 6-a);others carry point mutations resulting in amino acid substitutions, andas a consequence, they are more difficult to observe, though clearlypresent, in the hypervariable domains of env (cf. FIGS. 6-g and -h). Asnoted for point mutations, env gene and orf F also appear as moresusceptible to that form of genetic variation than the rest of thegenome. The degree of conservation of these repeats must be related totheir date of occurrence in the analyzed sequences: the moredegenerated, the more ancient. A very recent divergence of LAV_(BRU) andHTLV-3 is suggested by the extremely low number of mismatched AA betweentheir homologous proteins. However, one of the LAV_(BRU) repeats(located in the Hy1domain of env, FIG. 6-f) is not present in HTLV-3,indicating that this generation of tandem repeats is a rapid source ofgenetic diversity. We have found no traces of such a phenomenon, evenwhen comparing very closely related viruses, such as the Mason-Pfizermonkey virus (hereinafter “MPMV”) [Sonigo et al., 1986], and animmunosuppressive simian virus (hereinafter “SRV-1”) [Power et al.,1986]. Insertion or deletion of one copy of a direct repeat have beenoccasionally reported in mutant retroviruses [Shimotohno and Temin,1981; Darlix, 1986], but the extent to which we observe this phenomenonis unprecedented. The molecular basis of these duplications is unclear,but could be the “copy-choice” phenomenon, resulting from the diploidyof the retroviral genome [Varmus and Swanstrom, 1984; Clark and Mak,1983]. During the synthesis of the first-strand of the viral DNA, jumpsare known to occur from one RNA molecule to another, especially when abreak or a stable secondary structure is present on the template; aninaccurate re-initiation on the other RNA template could result in thegeneration (or the elimination) of a short direct repeat.

Genetic variability and subsequent antigenic modifications have oftenbeen developed by micro-organisms as a means for avoiding the host'simmune response, either by modifying their epitopes during the course ofthe infection, as in trypanosomes [Borst and Cross, 1982], or bygenerating a large repertoire of antigens, as observed in influenzavirus [Webster et al., 1982]. As the human AIDS virus is related toanimal lentiviruses [Sonigo et al., 1985; Chiu et al., 1985], itsgenetic variability could be a source of antigenic variation, as can beobserved during the course of the infection by the ovine lentivirusvisna [Scott et al., 1979; Clements et al., 1980] or by the equineinfectious anemia virus (hereinafter “EIAV”) [Montelaro et al., 1984].However, a major discrepancy with these animal models is the extremelylow, and possibly nonexistant, neutralizing activity of the sera ofindividuals infected by the AIDS virus, whether they are healthycarriers, displaying minor symptoms, or afflicted with AIDS [Weiss etal., 1985; Clave1 et al., 1985]. Furthermore, even for the visna virusthe exact role of antigenic variation in the pathogenesis is unclear[Thormar et al., 1983; Lutley et al., 1983]. We rather believe thatgenetic variation represents a general selective advantage forlentiviruses by allowing an adaptation to different environments, forexample by modifying their tissue or host tropisms. In the particularcase of the AIDS virus, rapid genetic variations are tolerated,especially in the envelope. This could allow the virus to become adaptedto different “micro-environments” of the membrane of their principaltarget cells, namely the T4 lymphocytes. These “micro-environments”could result from the immediate vicinity of the virus receptor topolymorphic surface proteins, differing either between individuals orbetween clones of lymphocytes.

Conserved Domains in the AIDS Virus Envelope

Since the proteins of most of the isolates are antigenicallycross-reactive, the genotypic differences do not seem to affect thesensitivity of actual diagnostic tests, based upon the detection ofantibodies to the AIDS virus and using purified virions as antigens.They nevertheless have to be considered for the development of the“second-generation” tests, that are expected to be more specific, andwill use smaller synthetic or genetically-engineered viral antigens. Theidentification of conserved domains in the highly immunogenic envelopeglycoprotein and the core structural proteins (gag) is very importantfor these tests. The conserved stretch found at the end of the OMP andthe beginning of the TMP (490–620, FIG. 3) could be a good candidate,since a bacterial fusion protein containing this domain waswell-detected by AIDS patients' sera [Chang et al., 1985].

The envelope, specifically the OMP, mediates the interaction between aretrovirus and its specific cellular receptor [DeLarco and Todaro, 1976;Robinson et al., 1980]. In the case of the AIDS virus, in vitro bindingassays have shown the interaction of the envelope glycoprotein gp110with the T4 cellular surface antigen [McDougal et al., 1986], alreadythought to be closely associated with the virus receptor [Klatzmann etal., 1984; Dagleish et al., 1984]. Identification of the AIDS virusenvelope domains that are responsible for this interaction(receptor-binding domains) appears to be fundamental for understandingof the host-viral interactions and for designing a protective vaccine,since an immune response against these epitopes could possibly elicitneutralizing antibodies. As the AIDS virus receptor is at least partlyformed of a constant structure, the T4 antigen, the binding site of theenvelope is unlikely to be exclusively encoded by domains undergoingdrastic genetic changes between isolates, even if these could beimplicated in some kind of an “adaptation”. One or several of theconserved domains of the OMP (residues 37–130, 211–289, and 488–530 ofFIG. 3 alignment), b:ought together by the folding of the protein, mustplay a part in the virus-receptor interaction, and this can be exploredwith synthetic or genetically-engineered peptides derived from thesedomains, either by direct binding assays or indirectly by assaying theneutralizing activity of specific antibodies raised against them.

African AIDS Viruses

Zaire and the neighboring countries of Central Africa are considered asan area endemic with the AIDS virus infection, and the possibility thatthe virus has emerged in Africa has became a subject of intensecontroversy (see Norman, 1985). From the present study, it is clear thatthe genetic organization of Zairian isolates is the same as that ofamerican isolates, thereby indicating a common origin. The veryimportant sequence differences observed between the proteins areconsistent with a divergent evolutionary process. In addition, the twoAfrican isolates are mutually more divergent than the American isolatesalready analyzed as far as that observation can be extrapolated, itsuggests a longer evolution of the virus in Africa and is alsoconsistent with the fact that a larger fraction of the population isexposed than in developed countries.

A novel human retrovirus with morphology and biologocal properties(cytopathogenicity, T4 tropism) similar to those of LAV, butnevertheless clearly genetically and antigenically distinct from it, wasrecently isolated from two patients with AIDS originating from GuineaBissau, West-Africa [Clavel et al., 1986]. In neighboring Senegal, thepopulation was seemingly exposed to a retrovirus also distinct from LAVbut apparently non-pathogenic [Barin et al., 1985; Kanki et al., 1986].Both of these novel African retroviruses seem to be antigenicallyrelated to the simian T-cell lymphotropic virus (hereinafter “STLV-III”)shown to be widely present in healthy African green monkeys and othersimian species [Kanki et al. 1985]. This raises the possibility of alarge group of African primate lentiviruses, ranging from the apparentlynon-pathogenic simian viruses to the LAV-type viruses. Their preciserelationship will only be known after their complete geneticcharacterization, but it is already very likely that they have evolvedfrom a common progenitor. The important genetic variability we haveobserved between isolates of the AIDS virus in Central Africa isprobably a hallmark of this entire group and may account for theapparently important genetic divergence between its members (loss ofcross-antigenicity in the envelopes). In this sense, the conservation ofthe tropism for the T4 lymphocytes suggests that it is a major advantageaquired by these retroviruses.

EXPERIMENTAL PROCEDURES

Virus Isolation

LAV_(ELI) was isolated from the peripheral blood lymphocytes of thepatient as described [Barre-Sinoussi et al., 1983]. Briefly, thelymphocytes were fractionated and co-cultivated withphytohaemagglutinin-stimulated normal human lymphocytes in the presenceof interleukin 2 and anti-alpha interferon serum. Viral production wasassessed by cell-free reverse transcriptase (hereinafter “RT”) activityassay in the cultures and by electron microscopy.

Molecular Cloning

Normal donor lymphocytes were acutely infected (10⁴ cpm of RTactivity/10⁶ cells) as described [Barré-Sinoussi et al., 1983], andtotal DNA was extracted at the beginning of the RT activity peak. Alambda library using the L47-1 vector [Loenen and Brammar, 1982] wasconstructed by partial HindIII digestion of the DNA as already described[Alizon et al., 1984]. About 5.10⁵ plaques for LAV_(ELI), obtained by invitro packaging (Amersham), were plated on E. coli LA101 and screened insitu under stringent conditions, using the 9 kb SacI insert of the clonelambda J19 [Alizon et al., 1984] carrying most of the LAV_(BRU) genomeas probe. Clones displaying positive signals were plaque-purified andpropagated on E. coli C600 recBC, and the recombinant phage E-H12carrying the complete genetic information of LAV_(ELI) was furthercharacterized by restriction mapping.

Nucleotide Sequence Strategy

Viral fragments derived from E-H12 were sequenced by the dideoxy chainterminator procedure [Sanger et al., 1977] after “shotgun” cloning inthe M13mp8 vector [Messing and Viera, 1982] as previously described[Sonigo et al., 1985]. The viral genome of LAV_(ELI) is 9176 nucleotideslong as shown in FIGS. 7A–7I. Each nucleotide of LAV_(ELI) wasdetermined from more than 5 independent clones on average.

Significance of the Figures

FIG. 1 contains an analysis of AIDS virus isolates, showing:

A/Restriction maps of the inserts of phage lambda clones derived fromcells infected with LAV_(ELI) (E-H12) and with LAV_(MAL) (hereinfter“M-H11”). The schematic genetic organization of the AIDS virus has beendrawn above the maps. The LTRs are indicated by solid boxes. Restrictionsites are indicated as follows: A:Aval; B:BamHI; Bg:BgIII; E:EcoRI;H:HindIII; Hc:HincII; K:KpnI; N:NdeI; P:PstI; S:SacI; and X:XbaI.

Asterisks indicate the HindIII cloning sites in lambda L47-1 vector.

B/A comparison of the sites for seven restriction enzymes in sixisolates: the prototype AIDS virus LAV_(BRU), LAV_(MAL) and LAV_(ELI);and Z1, Z2 and Z3. Restriction sites are represented by the followingsymbols vertically aligned wih the symbols in FIG. 1 a: ●:BgIII;★:EcoRI; ∇:HincII; ▾:HindIII; ♦:KpnI; ⋄:NdeI; and ∘:SacI.

FIG. 2 shows the genetic organization of the central region in AIDSvirus isolates. Stop codons in each phase are represented as verticalbars. Vertical arrows indicate possible AUG initiation codons. Spliceacceptor (A) and donor (D) sites identified in subgenomic viral mRNA[Muesing et al., 1985] are shown below the graphic of LAV_(BRU), andcorresponding sites in LAV_(ELI) and LAV_(MAL) are indicated. PPTindicates the repeat of the polypurine tract flanking the 3′LTR. Asobserved in LAV_(BRU) [Wain-Hobson et al., 1985], the PPT is repeated256 nucleotides 5′ to the end of the pol gene in both the LAV_(ELI) andLAV_(MAL) sequences, but this repeat is degenerated at two positions inLAV_(ELI).FIG. 3 shows an alignment of the protein sequences of four AIDS virusisolates. Isolate LAV_(BRU) [Wain-Hobson et al., 1985] is taken asreference; only differences with LAV_(BRU) are noted for ARV 2[Sanchez-Pescador et al., 1985] and the two Zairian isolates LAV_(MAL)and LAV_(ELI). A minimal number of gaps (−) were introduced in thealignments. The NH₂-termini of p₂₅ ^(gag) and p18^(gag) are indicated[Sanchez-Pescador, 1985]. The potential cleavage sites in the envelopeprecursor [Allan et al., 1985a; diMarzoVeronese, 1985] separating thesignal peptide (hereinafter “SP”), OMP and TMP are indicated as verticalarrows; conserved cysteines are indicated by black circles and variablecysteines are boxed. The one letter code for each amino acid is asfollows: A:Ala; C:Cys; D:Asp; E:Glu; F:Phe; G:Gly; H:His; I:Ile; K:Lys;L:Leu; M:Met; N:Asn; P:Pro; Q:Gln; R:Arg; S:Ser; T:Thr; V:Val; W:Trp;Y:Tyr.FIG. 4 shows a quantitation of the sequence divergence betweenhomologous proteins of different isolates. Part A of each table givesresults deduced from two-by-two alignments using the proteins ofLAV_(BRU) as reference, part B, those of LAV_(ELI) as reference.Sources: Muesing et al., 1985 for HTLV-3; Sanchez-Pescador et al., 1985for ARV 2 and Wain-Hobson et al., 1985 for LAV_(BRU). For each case inthe tables, the size in amino acids of the protein (calculated from thefirst methionine residue or from the beginning of the orf for pol) isgiven at the upper left part. Below are given the number of deletions(left) and insertions (right) necessary for the alignment. The largenumbers in bold face represent the percentage of amino acidssubstitutions (insertions/deletions being excluded). Two by twoalignments were done with computer assistance [Wilburg and Lipman,1983], using a gag penalty of 1, K-tuple of 1, and window of 20, exceptfor the hypervariable domains of env, where the number of gaps was mademinimum, and which are essentially aligned as shown in FIG. 3. Thesequence of the predicted protein encoded by orf R of HTLV-3 has notbeen compared because of a premature termination relative to all otherisolates.FIG. 5 shows the variability of the AIDS virus envelope protein. Foreach position x of the alignment of env (FIG. 3), variability V(x) wascalculated as: V(x)=number of different amino-acids at positionx/frequency of the most abundant amino acid at position x. Gaps in thealignments are considered as another amino acid. For an alignment of 4proteins, V(x) ranges from 1 (identical AA in the 4 sequences) to 16 (4different AA). This type of representation has previously been used in acompilation of the AA sequence of immunoglobulins variable regions [Wuand Kabat, 1970]. Vertical arrows indicate the cleavage sites; asterisksrepresent potential N-glysosylation sites (N-X-S/T) conserved in allfour isolates; black triangles represent conserved cysteine residues.Black lozanges mark the three major hydrophobic domains: OMP, TMP andSP; and the hyper-variable domains: Hyl, 2 and 3.FIG. 6 shows the direct repeats in the proteins of different AIDS virusisolates. These examples are derived from the aligned sequences of gag(a, b), F (c, d) and env (e, f, g, h) shown in FIG. 3. The two elementsof the direct repeat are boxed, while degenerated positions areunderlined.FIGS. 7A–7I show the complete cDNA sequence of LAV_(ELI) of thisinvention.

The invention thus pertains more specifically to the proteins,glycoproteins and other polypeptides including the polypeptidicstructures shown in the FIGS. 1–7. The first and last amino acidresidues of these proteins, glycoproteins and polypeptides carry numberscomputed from a first amino acid of the open-reading frames concerned,although these numbers do not correspond exactly to those of theLAV_(ELI) proteins concerned, rather to the corresponding proteins ofthe LAV_(BRU) or sequences shown in FIGS. 3A, 3B and 3C. Thus a numbercorresponding to a “first amino acid residue” of a LAV_(ELI) proteincorresponds to the number of the first amino-acyl residue of thecorresponding LAV_(BRU) protein which, in any of FIG. 3A, 3B or 3C, isin direct alignment with the corresponding first amino acid of theLAV_(ELI) protein. Thus the sequences concerned can be read from FIGS.7A–7I to the extent where they do not appear with sufficient clarityfrom FIGS. 3A–3F.

The preferred protein sequences of this invention extend between thecorresponding “first” and “last” amino acid residues. Also preferred arethe protein(s)- or glycoprotein(s)-portions including part of thesequences which follow:

OMP or gp110 proteins, including precursors:

-   -   1 to 530

OMP or gp110 without precursor:

-   -   34–530

Sequence carrying the TMP or gp41 protein:

-   -   531–877, particularly    -   680–700        well conserved stretches of OMP:    -   37–130,    -   211–289 and    -   488–530        well conserved stretch found at the end of the OMP and the        beginning of TMP:    -   490–620.

Proteins containing or consisting of the “well conserved stretches” areof particular interest for the production of immunogenic compositionsand (preferably in relation to the stretches of the env protein) ofvaccine compositions against the LAV-1 viruses.

The invention concerns more particularly all the DNA fragments whichhave been more specifically referred to in the drawings and whichcorrespond to open reading frames. It will be understood that oneskilled in the art will be able to obtain them all, for instance bycleaving an entire DNA corresponding to the complete genome ofLAV_(ELI), such as by cleavage by a partial or complete digestionthereof with a suitable restriction enzyme and by the subsequentrecovery of the relevant fragments. The DNA disclosed above can beresorted to also as a source of suitable fragments. The techniquesdisclosed in PCT application for the isolation of the fragments whichcan then be included in suitable plasmids are applicable here too. Ofcourse, other methods can be used, some of which have been examplifiedin European Application No. 178,978, filed Sep. 17, 1985. Reference isfor instance made to the following methods:

a) DNA can be transfected into mammalian cells with appropriateselection markers by a variety of techniques, such as calcium phosphateprecipitation, polyethylene glycol, protoplast-fusion, etc.

b) DNA fragments corresponding to genes can be cloned into expressionvectors for E. coli, yeast- or mammalian cells and the resultantproteins purified.

c) The provival DNA can be “shot-gunned” (fragmented) into procaryoticexpression vectors to generate fusion polypeptides. Recombinants,producing antigenically competent fusion proteins, can be identified bysimply screening the recombinants with antibodies against LAV antigens.

The invention further refers to DNA recombinants, particularly modifiedvectors, including any of the preceding DNA sequences adapted totransform corresponding microorganisms or cells, particularly eucaryoticcells such as yeasts, for instance Saccharomyces cerevisiae, or highereucaryotic cells, particularly cells of mammals, and to permitexpression of said DNA sequences in the corresponding microorganisms orcells. General methods of that type have been recalled in the abovesaidPCT international patent aplication PCT/EP 85/00548, filed Oct. 18,1985.

More particularly the invention relates to such modified DNA recombinantvectors modified by the abovesaid DNA sequences and which are capable oftransforming higher eucaryotic cells particularly mammalian cells.Preferably, any of the abovesaid sequences are placed under the directcontrol of a promoter contained in said vectors and recognized by thepolymerases of said cells, such that the first nucleotide codonsexpressed correspond to the first triplets of the above-defined DNAsequences. Accordingly, this invention also relates to the correspondingDNA fragments which can be obtained from the genome of LAV_(ELI) or itscDNA by any appropriate method. For instance, such a method comprisescleaving said LAV_(ELI) genome or its cDNA by restriction enzymespreferably at the level of restriction sites surrounding said fragmentsand close to the opposite extremities respectively thereof, recoveringand identifying the fragments sought according to sizes, if need bechecking their restriction maps or nucleotide sequences (or by reactionwith monoclonal antibodies specifically directed against epitopescarried by the polypeptides encoded by said DNA fragments), and furtherif need be, trimming the extremities of the fragment, for instance by anexonucleolytic enzyme such as Bal31, for the purpose of controlling thedesired nucleotid-sequences of the extremities of said DNA fragments or,conversely, repairing said extremities with Klenow enzyme and possiblyligating the latter to synthetic polynucleotide fragments designed topermit the reconstitution of the nucleotide extremities of saidfragments. Those fragments may then be inserted in any of said vectorsfor causing the expression of the corresponding polypeptide by the celltransformed therewith. The corresponding polypeptide can then berecovered from the transformed cells, if need be after lysis thereof,and purified by methods such as electrophoresis. Needless to say, allconventional methods for performing these operations can be resorted to.

The invention also relates more specifically to cloned probes which canbe made starting from any DNA fragment according to this invention, thusto recombinant DNAs containing such fragments, particularly any plasmidsamplifiable in procaryotic or eucaryotic cells and carrying saidfragments. Using the cloned DNA fragments as a molecular hybridizationprobe—either by labelling with radionucleotides or with fluorescentreagents—LAV virion RNA may be detected directly in the blood, bodyfluids and blood products (e.g. of the antihemophylic factors such asFactor VIII concentrates) and vaccines (e.g., hepatitis B vaccine). Ithas already been shown that whole virus can be detected in culturesupernatants of LAV producing cells. A suitable method for achievingthat detection comprises immobilizing virus on a support (e.g., anitrocellulose filter), disrupting the virion and hybridizing withlabelled (radiolabelled or “cold” fluorescent- or enzyme-labelled)probes. Such an approach has already been developed for Hepatitis Bvirus in peripheral blood [SCOTTO J. et al. Hepatology (1983), 3,379–384].

Probes according to the invention can also be used for rapid screeningof genomic DNA derived from the tissue of patients with LAV relatedsymptoms, to see if the proviral DNA or RNA present in host tissue andother tissues can be related to that of LAV_(ELI).

A method which can be used for such screening comprises the followingsteps: extraction of DNA from tissue, restriction enzyme cleavage ofsaid DNA, electrophoresis of the fragments and Southern blotting ofgenomic DNA from tissues, subsequent hybridization with labelled clonedLAV proviral DNA. Hybridization in situ can also be used.

Lymphatic fluids and tissues and other non-lymphatic tissues of humans,primates and other mammalian species can also be screened to see ifother evolutionnary related retrovirus exist. The methods referred tohereinabove can be used, although hybridization and washings would bedone under non-stringent conditions.

The DNAs or DNA fragments according to the invention can be used alsofor achieving the expression of viral antigens of LAV_(ELI) fordiagnostic purposes.

The invention relates generally to the polypeptides themselves, whethersynthesized chemically, isolated from viral preparations or expressed bythe different DNAs of the invention, particularly by the ORFs orfragments thereof in appropriate hosts, particularly procaryotic oreucaryotic hosts, after transformation thereof with a suitable vectorpreviously modified by the corresponding DNAs.

More generally, the invention also relates to any of the polypeptidefragments (or molecules, particularly glycoproteins having the samepolypeptidic backbone as the polypeptides mentioned hereinabove) bearingan epitope characteristic of a protein or glycoprotein of LAV_(ELI),which polypeptide or molecule then has N-terminal and C-terminalextremities respectively either free or, independently from each other,covalently bonded to amino acids other than those which are normallyassociated with them in the larger polypeptides or glycoproteins of theLAV virus, which last mentioned amino acids are then free or belong toanother polypeptidic sequence. Particularly, the invention relates tohybrid polypeptides containing any of the epitope-bearing-polypeptideswhich have been defined more specifically hereinabove, recombined withother polypeptides fragments normally foreign to the LAV proteins,having sizes sufficient to provide for an increased immunogenicity ofthe epitope-bearing-polypeptide yet, said foreign polypeptide fragmentseither being immunogenically inert or not interfering with theimmunogenic properties of the epitope-bearing-polypeptide.

Such hybrid polypeptides, which may contain from 5 up to 150, even 250amino acids, usually consist of the expression products of a vectorwhich contained ab initio a nucleic acid sequence expressible under thecontrol of a suitable promoter or replicon in a suitable host, whichnucleic acid sequence had however beforehand been modified by insertiontherein of a DNA sequence encoding said epitope-bearing-polypeptide.

Said epitope-bearing-polypeptides, particularly those whose N-terminaland C-terminal amino acids are free, are also accessible by chemicalsynthesis according to technics well known in the chemistry of proteins.

The synthesis of peptides in homogeneous solution and in solid phase iswell known. In this respect, recourse may be had to the method ofsynthesis in homogeneous solution described by Houbenweyl in the workentitled “Methoden der Organischen Chemie” (Methods of OrganicChemistry) edited by E. WUNSCH., vol. 15-I and II, THIEME, Stuttgart1974. This method of synthesis consists of successively condensingeither the successive amino acids in twos, in the appropriate order orsuccessive peptide fragments previously available or formed andcontaining already several amino-acyl residues in the appropriate orderrespectively. Except for the carboxyl and aminogroups which will beengaged in the formation of the peptide bonds, care must be taken toprotect beforehand all other reactive groups borne by these amino-acylgroups or fragments. However, prior to the formation of the peptidebonds, the carboxyl groups are advantageously activated, according tomethods well known in the synthesis of peptides. Alternatively, recoursemay be had to coupling reactions bringing into play conventionalcoupling reagents, for instance of the carbodiimide type, such as1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide. When the amino acidgroup used carries an additional amine group (e.g., lysine) or anotheracid function (e.g., glutamic acid), these groups may be protected bycarbobenzoxy or t-butyloxycarbonyl groups, as regards the amine groups,or by t-butylester groups, as regards the carboxylic groups. Similarprocedures are available for the protection of other reactive groups,for example, an —SH group (e.g., in cysteine) can be protected by anacetamidomethyl or paramethoxybenzyl group.

In the case of a progressive synthesis, amino acid by amino acid, thesynthesis starts preferably with the condensation of the C-terminalamino acid with the amino acid which corresponds to the neighboringaminoacyl group in the desired sequence and so on, step by step, up tothe N-terminal amino acid. Another preferred technique which can be usedis that described by R. D. Merrifield in “Solid Phase Peptide Synthesis”[J. Am. Chem. Soc., 45, 2149–2154]. In accordance with the Merrifieldprocess, the first C-terminal amino acid of the chain is fixed to asuitable porous polymeric resin, by means of its carboxylic group, theamino group of the amino acid then being protected, for example by at-butyloxycarbonyl group. When the first C-terminal amino acid is thusfixed to the resin, the protective group of the amine group is removedby washing the resin with an acid, i.e., trifluoroacetic acid, when theprotective group of the amine group is a t-butyloxycarbonyl group. Then,the carboxylic group of the second amino acid, which is to provide thesecond amino-acyl group of the desired peptidic sequence, is coupled tothe deprotected amine group of the C-terminal amino acid fixed to theresin. Preferably, the carboxyl group of this second amino acid has beenactivated, for example by dicyclohexyl-carbodiimide, while its aminegroup has been protected, for example by a t-butyloxycarbonyl group. Thefirst part of the desired peptide chain, which comprises the first twoamino acids, is thus obtained. As previously, the amine group is thende-protected, and one can further proceed with the fixing of the nextamino-acyl group and so forth until the whole peptide sought isobtained. The protective groups of the different side groups, if any, ofthe peptide chain so formed can then be removed. The peptide sought canthen be detached from the resin, for example by means of hydrofluoricacid, and finally recovered in pure form from the acid solutionaccording to conventional procedures.

As regards the peptide sequences of smallest size bearing an epitope orimmunogenic determinant, and more particularly those which are readilyaccessible by chemical synthesis, it may be requited, in order toincrease their in vivo immunogenic character, to couple or “conjugate”them covalently to a physiologically acceptable and non-toxic carriermolecule. By way of examples of carrier molecules or macromolecularsupports which can be used for making the conjugates according to theinvention can be mentioned natural proteins, such as tetanic toxoid,ovalbumin, serum-albumins, hemocyanins, etc. Synthetic macromolecularcarriers, for example polysines or poly(D-L-alanine)-poly(L-lysine)s,can be used too. Other types of macromolecular carriers that can beused, which generally have molecular weights higher than 20,000, areknown from the literature. The conjugates can be synthesized by knownprocesses such as are described by Frantz and Robertson in “Infect. andImmunity”, 33, 193–198 (1981) and by P. E. Kauffman in “Applied andEnvironmental Microbiology”, October 1981 Vol. 42, No. 4, pp. 611–614.For instance, the following coupling agents can be used: glutaricaldehyde, ethyl chloroformate, water-soluble carbodiimides suchas(N-ethyl-N′(3-dimethylamino-propyl) carbodiimide, HCl), diisocyanates,bis-diazobenzidine, di- and trichloro-s-triazines, cyanogen bromides andbenzaquinone, as well as the coupling agents mentioned in “Scand. J.Immunol.”, 1978, vol. 8, pp. 7–23 (Avrameas, Ternynck, Guesdon).

Any coupling process can be used for bonding one or several reactivegroups of the peptide, on the one hand, and one or several reactivegroups of the carrier, on the other hand. Again coupling isadvantageously achieved between carboxyl and amine groups carried by thepeptide and the carrier or vice-versa in the presence of a couplingagent of the type used in protein synthesis, e.g.,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxybenzotriazole,etc. Coupling between amine groups respectively borne by the peptide andthe carrier can also be made with glutaraldehyde, for instance,according to the method described by BOQUET, P. et al. (1982) Molec.Immunol., 19, 1441–1549, when the carrier is hemocyanin.

The immunogenicity of epitope-bearing-peptides can also be reinforced byoligomerisation thereof, for example in the presence of glutaraldehydeor any other suitable coupling agent. In particular, the inventionrelates to the water soluble immunogenic oligomers thus obtained,comprising particularly from 2 to 10 monomer units.

The glycoproteins, proteins and other polypeptides (generally designatedhereinafter as “antigens” of this invention) whether obtained bymethods, such as are disclosed in the earlier patent applicationsreferred to above, in a purified state from LAV_(ELI) virus preparationsor—as concerns more particularly the peptides by chemical synthesis, areuseful in processes for the detection of the presence of anti-LAVantibodies in biological media, particularly biological fluids such assera from man or animal, particularly with a view of possibly diagnosingLAS or AIDS.

Particularly the invention relates to an in vitro process of diagnosismaking use of an envelope glycoprotein or of a polypeptide bearing anepitope of this glycoprotein of LAV_(ELI) for the detection of anti-LAVantibodies in the serums of persons who carry them. Otherpolypeptides—particular those carrying an epitope of a core protein—canbe used too.

A preferred embodiment of the process of the invention comprises:

-   -   depositing a predetermined amount of one or several of said        antigens in the cups of a titration microplate;    -   introducing increasing dilutions of the biological fluid, to be        diagnosed (e.g., blood serum, spinal fluid, lymphatic fluid, and        cephalo-rachidian fluid), into these cups;    -   incubating the microplate;    -   washing carefully the microplate with an appropriate buffer;    -   adding into the cups specific labelled antibodies directed        against blood immunoglobulins and    -   detecting the antigen-antibody-complex formed, which is then        indicative of the presence of LAV antibodies in the biological        fluid.

Advantageously the labelling of the anti-immunoglobulin antibodies isachieved by an enzyme selected from among those which are capable ofhydrolysing a substrate, which substrate undergoes a modification of itsradiation-absorption, at least within a predetermined band ofwavelenghts. The detection of the substrate, preferably comparativelywith respect to a control, then provides a measurement of the potentialrisks, or of the effective presence, of the disease.

Thus, preferred methods of immuno-enzymatic and also immunofluorescentdetections, in particular according to the ELISA technique, areprovided. Titrations may be determinations by immunofluorescence ordirect or indirect immuno-enzymatic determinations. Quantitativetitrations of antibodies on the serums studied can be made.

The invention also relates to the diagnostic kits themselves for the invitro detection of antibodies against the LAV virus, which kits compriseany of the polypeptides identified herein and all the biological andchemical reagents, as well as equipment, necessary for peformingdiagnostic assays. Preferred kits comprise all reagents required forcarrying out ELISA assays. Thus preferred kits will include, in additionto any of said polypeptides, suitable buffers and anti-humanimmunoglobulins, which anti-human immunoglobulins are labelled either byan immunofluorescent molecule or by an enzyme. In the last instance,preferred kits also comprise a substrate hydrolysable by the enzyme andproviding a signal, particularly modified absorption of a radiation, atleast in a determined wavelength, which signal is then indicative of thepresence of antibody in the biological fluid to be assayed with saidkit.

It can of course be of advantage to use several proteins or polypeptidesnot only of LAV_(ELI), but also of LAV_(MAL) together with homologousproteins or polypeptides of earlier described viruses, such asLAV_(BRU), HTLV-3, ARV 2, etc.

The invention also relates to vaccine compositions whose activeprinciple is to be constituted by any of the antigens, i.e., thehereinabove disclosed polypeptides of LAV_(ELI), particularly thepurified gp110 or immunogenic fragments thereof, fusion polypeptides oroligopeptides in association with a suitable pharmaceutically orphysiologically acceptable carrier. A first type of preferred activeprinciple is the gp110 immunogen of said immunogens. Other preferredactive principles to be considered in that fields consist of thepeptides containing less than 250 amino acid units, preferably less than150, particularly from 5 to 150 amino acid residues, as deducible forthe complete genome of LAV_(ELI) and even more preferably those peptideswhich contain one or more groups selected from Asn-X-Thr and Asn-X-Seras defined above. Preferred peptides for use in the production ofvaccinating principles are peptides (a) to (f) as defined above. By wayof example, there may be mentioned that suitable dosages of the vaccinecompositions are those which are effective to elicit antibodies in vivo,in the host, particularly a human host. Suitable doses range from 10 to500 micrograms of polypeptide, protein or glycoprotein per kg, forinstance 50 to 100 micrograms per kg.

The different peptides according to this invention can also be usedthemselves for the production of antibodies, preferably monoclonalantibodies specific for the respective different peptides. For theproduction of hybridomas secreting said monoclonal antibodies,conventional production and screening methods can be used. Thesemonoclonal antibodies, which themselves are part of the invention,provide very useful tools for the identification and even determinationof relative proportions of the different polypeptides or proteins inbiological samples, particularly human samples containing LAV or relatedviruses.

The invention further relates to the hosts (procaryotic or eucaryoticcells) which are transformed by the above mentioned recombinants andwhich are capable of expressing said DNA fragments.

Finally the invention also concerns vectors for transforming eucaryoticcells of human origin, particularly lymphocytes, the polymerase of whichare capable of recognizing the LTRs of LAV. Particularly said vectorsare characterized by the presence of a LAV LTR therein, said LTR beingthen active as a promoter enabling the efficient transcription andtranslation in a suitable host of a DNA insert coding for a determinedprotein placed under its controls.

Needless to say, the invention extends to all variants of genomes andcorresponding DNA fragments (ORFS) having substantially equivalentproperties, all of said genomes belonging to retroviruses which can beconsidered as equivalents of LAV_(ELI). It must be understood that theclaims which follow are also intended to cover all equivalents of theproducts (glycoproteins, polypeptides, DNAs, etc.) whereby an equivalentis a product, e.g., a polypeptide, which may distinguish from a productdefined in any of said claims, say through one or several amino acids,while still having substantially the same immunological or immunogenicproperties. A similar rule of equivalency shall apply to the DNAs, itbeing understood that the rule of equivalency will then be tied to therule of equivalency pertaining to the polypeptides which they encode.

It will also be understood that all the literature referred tohereinbefore and hereinafter and all patent applications and patents notspecifically identified herein but which form counterparts of thosespecifically designated herein, must be considered as incorporatedherein by reference.

It should further be mentioned that the invention further relates toimmunogenic compositions that contain preferably one or more of thepolypeptides, which are specifically identified above and which have theamino acid sequences of LAV_(ELI) that have been identified, or peptidicsequences corresponding to previously defined LAV proteins. In thisrespect, the invention relates more particularly to the particularpolypeptides which have the sequences corresponding more specifically tothe LAV_(BRU) sequences which have been referred to earlier, i.e., thesequences extending between the following first and last amino acids, ofthe LAV_(BRU) proteins themselves, i.e., the polypeptides havingsequences contained in the LAV_(BRU) OMP or LAV_(BRU) TMP or sequencesextending over both, particularly those extending from between thefollowing positions of the amino acids included in the env open readingframe of the LAV_(BRU) genome,

-   -   1–530    -   34–530        and more preferably    -   531–877, particularly 680–700,    -   37–130    -   211–289    -   488–530    -   490–620.

These different sequences can be used for any of the above definedpurposes and in any of the compositions which have been disclosed.

Finally the invention also relates to the different antibodies which canbe formed specifically against the different peptides which have beendisclosed herein, particularly to the monoclonal antibodies whichrecognize them specifically. The corresponding hybridomas which can beformed starting from spleen cells previously immunized with suchpeptides which are fused with appropriate myeloma cells and selectedaccording to standard procedures also form part of the invention.

Phage λ clone E-H12 derived from LAV_(ELI) infected cells has beendeposited at the CNCM under No. I-550 on May 9, 1986. Phage clone M-H11derived from LAV_(MAL) infected cells has been deposited at the CNCMunder No. I-551 on May 9, 1986.

REFERENCES

-   Alizon, M., Sonigo, P., Barré-Sinoussi, F., Chermann, J. C.,    Tiollais, P., Montagnier, L. & Wain-Hobson, S. (1984). Molecular    cloning of lymphadenopathy-associated virus. Nature 312, 757–760.-   Allan, J. S., Coligan, J. E., Barin, F., McLane, M. F., Sodroski, J.    G., Rosen, C. A., Haseltine, W. A., Lee, T. H., & Essex, M. (1985a).    Major glycoprotein antigens that induce antibodies in AIDS patients.    Science 228, 1091–1094.-   Allan, J. S., Coligan, J. E., Lee, T. H., McLane, M. F., Kanki, P.    J., Groopman, J. E., & Essex, M. (1985b). A new HTLV-III/LAV antigen    detected by antibodies from AIDS patients. Science 230, 810–813.-   Arya, S. K., Guo, C., Josephs, S. F., & Wong-Staal, F. (1985).    Trans-activator gene of human T-lymphotropic virus type III    (HTLV-III). Science 229, 69–73.-   Bailey, A. C., Downing, R. G., Cheinsong-Popov, R., Tedder, R. C.,    Dalgleish, A. G., & Weiss, R. A. (1985). HTLV-III serology    distinguishes atypical and endemic Kaposi's sarcoma in Africa.    Lancet I, 359–361.-   Barin, F., M'Boup, S., Denis, F., Kanki, P., Allan, J. S., Lee, T.    M., & Essex, M. (1985). Serological evidence for virus related to    simian T lymphotropic retrovirus in residents of West Africa. Lancet    II, 1387–1389.-   Barré-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T.,    Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Brun-Vézinet,    F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. (1983). Isolation    of a T-lymphotropic retrovirus from a patient at risk of acquired    immune deficiency syndrome (AIDS). Science 220, 868–870.-   Been, S., Rutledge, R., Folks, T., Gold, J., Baker, L. McCormick, J.    Feorino, P., Piot, P., Quinn T. & Martin, M. (1985). Genomic    heterogeneity of AIDS retroviral isolates from North America and    Zaire. Science 230, 949–951.-   Borst, P., & Cross, G. A. M. (1982). Molecular basis for trypanosome    antigenic variation. Cell 29, 291–303.-   Brun-Vésinet, F., Rouzioux, C., Montagnier, L., Chamaret, S.,    Gruest, J., Barré-Sinoussi, F., Geroldi, D., Chermann, J. C.,    McCormick, J. Mitchell, S., Piot, P., Taelmann, H. Minlangu, K. B.,    Wobin, O., Mbendi, N. Mazebo, P., Kalambayi, K. Bridts, C.,    Desmyter, J., Feinsod, F., & Quinn T. C. (1984). Prevelance of    antibodies to lymphadenopathy-associated virus in African patients    with AIDS. Science 226, 453–456.-   Chang, N. T., Chanda, P. K., Barone, A. D., McKinney, S., Rhodes, D.    P., Tam, S. H., Shearman, C. W., Huang, J. & Chang, T. W. (1985).    Expression in Escheridia coli of open reading frame gene segments of    HTLV-III. Science 228, 93–96.-   Chiu, I. M., Yaniv, A., Dahlberg, J. E., Gazit, A., Skuntz, S. F.,    Tronick, S. R. & Aaronson, S. A. (1985). Nucleotide sequence    evidence for relationship of AIDS retrovirus to lentiviruses. Nature    317, 366–368.-   Clark, S. P., & Mak, T. W., (1984). Fluidity of a retrovirus    genome. J. Virol. 50, 759–765.-   Clavel, F., Klatzmann, D., & Montagnier, L., (1985). Deficient    neutralizing capacity of sera from patients with AIDS or related    syndromes. Lancet I, 879–880.-   Clavel, F., Brun-Vézinet, F., Guetard, D., Chamaret, S., Laurent,    A., Rouzioux, C., Rey, M., Katlama, C., Rey, F., Champelinaud, J.    L., Nina, J. S., Mansinho, K., Santos-Ferreira, M. O., Klatzmann,    D., & Montagnier, L. (1986). LAV type II: a second retrovirus    associated with AIDS in West-Africa. C.R. Acad. Sci. Paris 302,    485–488.-   Clements, J. E., Narayan, O., Griffin, D. E. and Johnson, R. T.    (1980). genomic changes associated with antigenic variation of visna    virus during persistent infection. Proc. Natl. Acad. Sci. USA 77,    4454–4458.-   Clumeck, N., Sonnet, J., Taelman, M., Mascart-Lemone, F., De    Bruyére, M., Van de Perre, P., Dasnoy, J., Marcelis, L., Lamy, M.,    Jonas, C., Eyckmans, L., Noel, H., Vanhaeverbeek, M. &    Butzler, J. P. (1984). Acquired immune deficiency syndrome in    African patients. N. Engl. J. Med., 10, 492–497.-   Dalgleish, A. G., Beverley, P. C., Clapham P. R., Crawford, D. H.,    Greaves, M. F. & Weiss, R. A. (1984). The CD4; (T4) antigen is an    essential component of the receptor for the AIDS retrovirus. Nature    312, 763–767.-   Darlix, J. L. (1986) Control of Rous sarcoma virus RNA translation    and packaging by the 5′ and 3′ untranslated sequences. J. Mol.    Biol., in the press.-   DeLarco, J. & Todaro, G. J. (1976). Membrane receptors of murine    leukemia viruses: characterization using the purified viral envelope    glycoprotein, gp71. Cell 8, 365–371.-   DiMarzoVeronese, F., DeVico, A. L., Copeland, T. D., Oroszlan, S.,    Gallo, R. C., & Sarngadharan, M. G. (1985). Characterization of gp    41 as the transmembrane protein coded by the HTLV-III/LAV envelope    gene. Science 229, 1403–1405.-   Ellrodt, A., Barré-Sinoussi, F., Le Bras, P., Nugeyre, M. T.,    Brun-Vézinet, F., Rouzioux, C., Segond, P., Caquet, R.,    Montagnier, L. & Chermann, J. C. (1984). Isolation of human    T-lymphotropic retrovirus (LAV) from Zairan married couple, one with    AIDS, one with prodromes. Lancet I, 1383–1385.-   Hahn, B. H., Shaw, G. M., Arya, S. U., Popovic, M., Gallo, R. C., &    Wong-Staal, F. (1984). Molecular cloning and characterizaion of the    HTLV-III virus associated with AIDS. Nature 312, 166–169.-   Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S., &    Van de Pol, S. (1982). Rapid evolution of RNA genomes. Science 215,    1577–1585.-   Kan, N. C., Franchini, G., Wong-Staal, F., Dubois, G. C., Robey, W.    G., Lautenberger, J. A., & Papas, T. S. (1986). Identification of    HTLV-III/LAV sor gene product and detection of antibodies in human    sera. Science 231, 1553–1555.-   Kanki, P. J., Alroy, J. & Essex, M. (1985). Isolation of    T-lymphotropic retroviruses from wild-caught African Green Monkeys.    Science 230, 951–954.-   Kanki, P. J., Barin, F., M'Boup, S., Allan, J. S., Romet-Lemonne, J.    L., Markink, R., McLane, M. F., Lee, T. H., Arbeille, B., Denis, F.    & Essex, M. (1986). New human T-lymphotropic retrovirus related to    simian T-lymphotropic virus type III (STLV-III ). Science, 232,    238–243.-   Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D.,    Hercend, T., Gluckman, J. C., & Montagnier, L. (1984). T-lymphocyte    T4 molecule behave as the receptor for human retrovirus LAV. Nature    312, 767–768.-   Kyte, J. & Doolittle, R., (1982). A simple methof for displaying the    hydropathic character of a protein. J. Mol. Biol. 157, 105–132.-   Koch, W., Hunsmann, G. & Friedrich, R. (1983). Nucleotide sequence    of the envelope gene of Friend murine leukemia virus. J. Virol., 45,    1–9.-   Lee, T. H., Coligan, J. E. Allan, J. S., McLane, M. F.,    Groopman, J. E. & Essex, M. (1986). A new HTLV III/LAV protein    encoded by a gene found in cytopathic retroviruses. Science 231,    1546–1549.-   Loenec, W. A. M. & Brammar, W. J. (1980). A bacteriophage lambda    vector for cloning large DNA fragments made with several restriction    enzymes. Gene 10, 249–259.-   Lutley, R., Petursson, G., Palsson, P. A., Georgsson, G., Klein, J.,    & Nathanson, N, (1983). Antigenic drift in visna: virus variation    during longterm infection of icelandic sheep. J. Gen. Virol. 64,    1433–1440.-   MacDougal, J. S., Kennedy, M. S., Sligh, J. M., Cort, S. P.,    Mawle, A. & Nicholson, J. K. A. (1986). Binding of HTLV-III/LAV to    T4⁺ cells by a complex of the 110 k viral protein and the T4    molecule. Science 231, 382–385.-   Messing, J. and Viera, J. (1982). A new pair of M13 vectors for    selecting either DNA strand of double digest restriction fragments.    Gene 19, 269–276.-   Montagnier, L. (1985). Lymphadenopathy-associated virus: from    molecular biology to pathogenicity. Ann. Inter. Med. 103, 689–693.-   Montagnier, L., Clavel, F., Krust, B., Chamaret, S., Rey, F.,    Barré-Sinoussi, F. & Chermann, J. C. (1985). Identification and    antigenicity of the major envelope glycoprotein of    lymphadenopathy-associated virus. Virology 144, 283–289.-   Montelaro, R. C., Parekh, B., Orrego, A. & Issel, C. J. (1984).    Antigenic variation during persistent infection by equine infectious    anemia virus, a retrovirus. J. Biol. Chem., 250, 10539–10544.-   Muesing, M. A., Smith, D. M., Cabradilla, C. D., Benton, C. V.,    Lasky, L. A. & Capon, D. J. (1985). Nucleic acid structure and    expression of the human AIDS/lymphadenopathy retroviruses. Nature    313, 450–458.-   Norman, C. (1985). Politics and science clash on African AIDS.    Science 230, 1140–1142.-   Piot, P., Quinn, T. C., Taelman, H., Feinsod, F. M., Minlangu, K.    B., Wobin, O., Mbendi, N., Mazebo, P., Ndongi, K., Stevens, W.,    Kalambayi, K., Mitchell, S., Bridts, C. & McCormick, J. B. (1984).    Acquired immunodeficiency syndrome in-   Power, M. D., Marx, P. A., Bryant, M. L., Gardner, M. B.,    Barr, P. J. & Luciw, P. A. (1986). Nucleotide sequence of SRV-1, a    type D simian acquired immune deficiency syndrome retrovirus.    Science 231, 1567–1572.-   Rabson, A. B. & Martin, M. A. (1985). Molecular organization of the    AIDS retrovirus. Cell 40, 477–480.-   Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B.,    Josephs, S. F., Doran, E. R., Rafalski, A., Whitchorn, E. A.,    Baumeister, K., Ivanoff, L., Petteway, S. R., Pearson, M. L.,    Lautenbergen, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T.,    Gallo, R. C. & Wong-Staal, F. (1985). Complete nucleotide sequence    of the AIDS virus, HTLV-III. Nature 313, 277–284.-   Robinson, P. J. G., Hunsmann, G., Schneider, J. & Schirrmacher, V.    (1980). Possible cell surface receptor for Friend murine leukemia    virus is isolated with viral envelop glycoprotein complexes. J.    Virol., 36, 291–294.-   Rosen, C. A., Sodroski, J. G. & Haseltine, W. A. (1985). The    location of cisacting regulatory sequences in the human T cell    lymphotropic virus type III (HTLV-III/LAV) long terminal repeat.    Cell 41, 813–823.-   Sanchez-Pescador, R. Power, M. D., Barr, P. J., Steimer, K. S.,    Stemfeieb, M. M., Brown-Shimer, S. L., Gee, W. W., Bernard, A.,    Randolph, A., Levy, J. A., Dina, D. & Luciw, P. A., (1985).    Nucleotide sequence and expression of an AIDS-associated retrovirus    (ARV-2). Science 227, 484–492.-   Sanger, F., Nicklen, S. & Coulsen, A. R. (1977). DNA sequencing with    chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,    5463–5467.-   Scott, J. V., Stowring, L., Haase, A. T., Narayan, O. & Vigne, R.    (1979). Antigenic variation in visna virus. Cell 18, 321–327.-   Shimotohno, K., & Temin, H. (1982). Spontaneous variation and    synthesis in the U3 region of the long terminal repeat of an avian    retrovirus. J. Virol. 41, 163–171.-   Sodroski, J., Patarca, R., Rosen C., Wong-Staal, F. & Haseltine, W.    (1985). Location of the trans-activating region of the genome of    human T-cell lymphotropic virus type II. Science 229, 74–77.-   Sonigo, P., Alizon, M., Staskus, K., Klatzmann, D., Cole, S., Danos,    O., Retzel, E., Tiollais, P., Haase, A. & Wain-Hobson, S. (1985).    Nucleotide sequence of the visna lentivirus: Relationship to the    AIDS virus. Cell 42, 369–382.-   Sonigo, P., Barker, C., Hunter, E. & Wain-Hobson S. (1986).    Nucleotide sequence of Mason-Pfizer Monkey virus: an    immunosuppressive D-type retrovirus. Cell, in the press.-   Steinhauer, D. A., & Holland, J. H. (1986). Direct method for    quantitation of extreme polymerase error frequencies at selected    single base in viral RNA. J. Virol. 57, 219–228.-   Thormar, H., Barshatsky, M. R., Arnesen, K., & Kozlowski, P. B.    (1983). The emergence of antigenic variants is a rare event in    long-term visna virus invention in vivo. J. Gen. Virol. 64,    1427–1432.-   Van de Perre, P., Rouvroy, D., Lepage, P., Bogaerts, J., Kestelyn,    P., Kayihigi, J., Hekker, A. C., Butzler, J. P. & Clumeck, N.    (1984). Acquired immunodeficiency syndrome in Rwanda. Lancet II,    62–65.-   Varmus, H. & Swanstrom, R. (1984). Replication of retroviruses. In    Molecular biology of the tumor viruses/RNA tumor viruses. R.    Weiss, N. Teich, H. Varmus, J. Coffin, eds. (Cold Spring Harbor    Laboratory, New York), vol. 1, pp. 369–512.-   Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S., & Alizon, M.    (1985). Nucleotide sequence of the AIDS virus, LAV. Cell 40, 9–17.-   Webster, R. G., Laver, W. G., Air, G. M. & Schild, G. C. (1982).    Molecular mechanisms of variation in influenza viruses. Nature 296,    115–121.-   Weiss, R. A. (1984). Human T-cell retroviruses. In Molecular biology    of the tumor viruses: RNA viruses. R. Weiss, N. Teich, H. Varmus, J.    Coffin, eds. (Cold Spring Harbor Laboratory, New York), vol II:    supplement, pp. 405–485.-   Weiss, R. A., Clapham, P. R., Cheinson-Popov, R. Dalgleish, A. G.,    Carne, C. A., Weller, I. A. D. & Tedder, R. C. (1985).    Neutralization of human T-lymphotropic virus type III by sera of    AIDS and AIDS-risk patients, Nature, 316, 69–72.-   Wilburg, W. J., & Lipman, D. J. (1983). Rapid similarity searches of    nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA 80,    726–730.-   Wu, T. T., & Kabat, E. A. (1970). An analysis of the sequences of    the variable regions of Bence-Jones proteins and myeloma light    chains and their implications for antibody complementarity. J. Exp.    Med. 132, 211–250.

1. A purified HIV-1 virus, wherein said virus encodes an HIV-1_(ELI)variant Env protein containing the well-conserved stretches of OMP atamino acid positions 37–130, 211–289, and 488–530 of HIV-1_(ELI) orHIV-1_(MAL) shown in FIG. 3E.
 2. The purified virus of claim 1, whereinsaid HIV-1_(ELI) variant Env protein has the following amino acidresidues at positions 37–130:LWVTVYYGVPVWKEATTTLFCASDAKSYETEAHNIWATHACVPTDPNPQEIALENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCV.
 3. The purified virus of claim1, wherein said HIV-1_(ELI) variant Env protein has the following aminoacid residues at positions 211–289:CNTSAITQACPKVSFEPIPIHYCAPAGFAILKCRDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVIIRS.
 4. The purified virus of claim 1, wherein saidHIV-1_(ELI) variant Env protein has the following amino acid residues atpositions 488–530: RPGGGDMRDNWRSELYKYKVVQIEPLGVAPTRAKRRVVEREKR.
 5. Thepurified virus of claim 1, wherein said HIV-1_(ELI) variant Env proteinhas the following amino acid residues at positions 37–130, 211–289, and488–530: LWVTVYYGVPVWKEATTTLFCASDAKSYETEAHNIWATHACVPTDPNPQEIALENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCV;CNTSAITQACPKVSFEPIPIHYCAPAGFAILKCRDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVIIRS; andRPGGGDMRDNWRSELYKYKVVQIEPLGVAPTRAKRRVVEREKR.